Perturb-seq identified novel target genes and molecular pathways regulating cardiac fibrosis

by Afifa Binta Saifuddin
Doctoral thesis
Date of Examination:2025-05-07
Date of issue:2025-06-12
Advisor:Prof. Dr. Elisabeth Zeisberg
Referee:Prof. Dr. André Fischer
Referee:Prof. Dr. Matthias Dobbelstein
Persistent Address: http://dx.doi.org/10.53846/goediss-11288

 

 

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Abstract

English

Cardiac fibrosis is a prevalent feature in all forms of chronic heart disease, for which no definitive remedy exists to date. A hallmark of fibrosis is the transition of fibroblasts into their activated form, known as myofibroblasts. The molecular mechanism underlying this transition remains largely unknown. In this study, we aimed to use Perturb-seq to gain a comprehensive understanding of the complexity of myofibroblast activation and to identify novel factors regulating cardiac fibrosis. The insights obtained from this work may aid in developing therapies for cardiac fibrosis. Perturb-seq technology combines single-cell RNA sequencing (scRNA-seq) with CRISPR screens, enabling the identification of both the genetic perturbation and transcriptomic profile at the single-cell level. We have generated a sgRNA library targeting more than ~2000 murine cardiac transcriptional regulators in an AAV9 mutant capable of effectively transducing Cas9+ primary mouse cardiac fibroblasts. scRNA-seq analysis of transduced cells, both with and without TGFβ stimulation, identified nine anti-fibrotic gene candidates: Arnt, Hoxc13, Jup, Foxo4, Csrp1, Dtx3, Ifngr2, Stk17b and Add3. Gene set enrichment analysis (GSEA) on scRNA-seq data shows significant enrichment of fibrosis and TGFβ-signaling-associated gene sets following knockdown of these gene candidates, even in the absence of TGFβ stimulation. We further validated the anti-fibrotic effect of Hoxc13, one of the gene candidates, in human fibroblast cell line. The silencing of the Hoxc13 gene in human fibroblasts increased the level of fibrotic markers such as Col1a1 and αSMA, while its overexpression drastically reduced the secretion of Cola1a1, indicating its potential role as an anti-fibrotic factor. In conclusion, our study is the first to apply Perturb-seq for a single-cell genetic screen to identify novel fibrotic regulators. These findings highlight its great potential in uncovering therapeutic targets for the management of cardiac fibrosis.
Keywords: Fibrosis; Novel transcriptional regulator; Perturb-seq
Schlagwörter: Fibrosis; Novel transcriptional regulator; Perturb-seq